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Pneumocystis carinii pneumonia (PCP) is one of the most predominant opportunistic infectious diseases in patients with AIDS. Nested PCR has been described as a sensitive and specific tool for detecting P. carinii DNA in clinical specimens. Little is known about the correlation of positive PCR results and clinical evidence of PCP in patients with different forms of immunosuppression. One hundred and thirty-six sputum samples, 26 tracheal-bronchial aspirate samples, 35 bronchoalveolar lavage samples, and 11 lung biopsy samples from (i) human immunodeficiency virus (HIV)-infected patients with AIDS, (ii) immunocompromised patients with leukemia or lymphoma, and (iii) immunocompetent control patients were investigated by a nested PCR amplifying DNA from the mitochondrial large subunit of P. carinii. All patients suffered from acute episodes of respiratory disease. The resulting data were correlated with clinical evidence of PCP. A high degree of association of positive P. carinii PCR results and clinical evidence of PCP in HIV-infected patients with AIDS was found. When calculated for bronchoalveolar lavage and lung biopsy samples, the positive and the negative predictive values of P. carinii PCR for PCP diagnosis in HIV-infected patients with AIDS were 1 and the specificity and the sensitivity were 100%. In contrast, in the group of patients with leukemia or lymphoma, the positive predictive value of the nested PCR for these materials was found to be as low as 0.09, the negative predictive value was 0.73, the specificity was 44.4%, and the sensitivity was 25.0%. No P. carinii DNA could be detected in specimens from immunocompetent patients. In summary, in contrast to patients with leukemia and lymphoma, nested PCR seems to be a sensitive and specific tool for PCP diagnosis in HIV-infected patients with AIDS.

To evaluate the value of single and nested PCRs for diagnosis of Pneumocystis carinii pneumonia (PCP) in a variety of respiratorily distressed patient groups, 574 respiratory samples from 334 patients (89 human immunodeficiency virus [HIV]-positive patients, 61 transplant recipients, 66 malignancy patients, 34 otherwise immunosuppressed patients, and 84 immunocompetent patients) were prospectively examined by microscopy and single and nested PCRs. The resulting data were correlated with clinical evidence of PCP. Microscopy and single PCR of bronchoalveolar lavage (BAL) specimens from HIV patients were 100% sensitive and specific in detecting PCP, whereas nested PCR, although being 100% sensitive, reached a specificity of only 97.5%. In the three non-HIV immunosuppressed patient groups, both single and nested PCR invariably produced lower positive predictive values than microscopy. Among immunocompetent patients, the positive predictive values of both PCRs were 0%. Therefore, the diagnostic values of the PCR methods tested do not seem to offer any additional advantage compared to that of conventional microscopy for these patient groups. However, nested PCR identified a significant percentage of clinically silent P. carinii colonizations in about 17 to 20% of immunocompetent and immunosuppressed non-HIV patients.

To evaluate the risk of a nosocomial spread of Pneumocystis carinii f. sp. hominis (P. carinii hominis), air filter samples from rooms of P. carinii pneumonia (PCP) patients, adjacent corridors, and other hospital environments have been investigated for the presence of P. carinii hominis. Amplified DNA from air filters and sputum or bronchoalveolar lavage samples from the PCP patients have been genotyped with the P. carinii hominis genes of the mitochondrial large-subunit (mtLSU) rRNA and the internal transcribed spacers (ITS1 and ITS2) of the rRNA. Genotypes of the two loci were identified by direct sequencing, and for site 85 of the mtLSU locus, three allele-specific PCR assays were used. P. carinii hominis DNA was identified in the air of five of seven PCP patient rooms and in the air of two of four air filtrations from the ward corridors. The P. carinii hominis genotypes were the same in four of the five room air samples as those in the corresponding patients, suggesting a risk of person-to-person transmission of P. carinii hominis from PCP patients. Three of 16 air samples collected in infectious disease wards without the presence of PCP patients and one sample from a cardiology unit in a separate hospital building were also positive, which further strengthens the possibility of acquisition of P. carinii hominis from the environment.

An enzyme-linked immunosorbent assay and a Western blot analysis were developed to study the antibody response to Pneumocystis carinii in serum and bronchoalveolar lavage fluid from 27 human immunodeficiency virus 27 (HIV)-infected patients with P. carinii pneumonia (Pcp), 32 patients without Pcp, and 51 HIV-negative controls. Urea was used for the correct dilution of epithelial lining fluid, and albumin was used to evaluate transudation from plasma for the assessment of local production of antibodies to P. carinii. By contrast with those of immunoglobulin G (IgG), IgA responses to P. carinii were increased in serum from HIV-positive patients compared to negative controls. Local production of antibodies to P. carinii, especially IgA, was decreased in patients with Pcp. In a study of 10 patients of each group, IgG and IgA responses to gp116 from P. carinii were lower in patients with Pcp than in other groups. These results suggest that, in addition to alveolar macrophages, local antibodies may play a role in host defense against P. carinii.

The objective of this study was to type, analyze, and compare Pneumocystis carinii hominis strains obtained from different samples during a given or recurrent episodes of P. carinii pneumonia (PCP) for epidemiologic purposes. We studied 36 bronchoalveolar lavage (BAL) or induced sputum (IS) samples from 16 human immunodeficiency virus-infected patients with one or several episodes of PCP. PCR amplification and direct sequencing were performed on the two internal transcribed spacers (ITS1 and ITS2) of P. carinii hominis rRNA genes by using DNA extracted from BAL or IS samples, and the sequences were compared to the mitochondrial large-subunit (mt LSU) gene sequence determined in a previous study in our laboratory. The studies of the mt LSU and ITS sequences showed that some patients (n = 10) were infected with the same strains of P. carinii hominis during a given episode of PCP. In one patient infected with strains with identical sequences in several episodes, the recurrence could have been due to reactivation of organisms not eliminated by treatment during the first episode or to de novo infection by an identical strain. In five patients infected with strains with different sequences in each episode, recurrence was due to de novo infection. Sequence analysis of these two P. carinii hominis gene regions showed that de novo infection can occur in AIDS patients with recurrent PCP.

AIM: To compare the results of DNA amplification by the polymerase chain reaction (PCR) with immunofluorescence staining for detecting Pneumocystis carinii in bronchoalveolar lavage specimens taken from symptomatic HIV seropositive patients with suspected P carinii pneumonia (PCP). METHODS: Bronchoalveolar lavage specimens were obtained from 28 symptomatic HIV seropositive patients. Specimens were examined for P carinii using immunofluorescence, and by DNA amplification with PCR to obtain results on gel electrophoresis (gel) and a more sensitive Southern hybridisation (blot) technique. Specimens positive by immunofluorescence and gel electrophoresis were serially diluted to a 10(-6) concentration and each dilution strength tested for P carinii using PCR to compare quantitatively immunofluorescence with PCR. RESULTS: Of the 28 specimens analysed, 18 were negative for P carinii by both immunofluorescence and PCR, two were positive only by the blot technique of PCR, four were equivocally positive and four unequivocally positive by immunofluorescence. Three of the four equivocally positive patients tested by immunofluorescence were negative for P carinii by PCR, although one was positive by PCR (blot) technique. This patient had clinically confirmed PCP. Of the four unequivocally positive patients tested by immunofluorescence, three were gel and blot positive by PCR and had PCP clinically, but one was negative by both gel and blot techniques, although the patient certainly had PCP on clinical grounds. This patient had received nine days of treatment with high dose co-trimoxazole before bronchoalveolar lavage specimens were obtained. The three specimens positive by gel and blot techniques remained gel positive down to dilutions of between 10(-4) and 10(-6). CONCLUSIONS: PCR results may become negative soon after starting treatment for PCP. Specimens should therefore be taken before, or soon after, starting treatment. PCR seems to be between 10(4) and 10(6) times more sensitive than immunofluorescence.

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The detection of Pneumocystis carinii DNA by PCR was compared with routine cytologic staining techniques (CYT). A total of 284 clinical respiratory specimens, including 137 bronchoalveolar lavage (BAL), 63 bronchoalveolar washing, 63 sputum, and 21 induced sputum samples, obtained from patients with or at high risk for human immunodeficiency virus infection were evaluated. Eighty specimens were positive by PCR, and 69 were positive by CYT. PCR was able to detect P. carinii in more bronchoalveolar washing specimens (15 versus 11) and in comparable BAL specimens (53 versus 54) compared with CYT. PCR was particularly more sensitive than CYT in detecting P. carinii in expectorated sputum (12 versus 4 samples). Of the 19 patients whose respiratory specimens were positive for P. carinii by PCR but negative by CYT, 5 had P. carinii pneumonia (PCP) confirmed by subsequent BAL and transbronchial or mediastinal lymph node biopsy and 9 had a clinical course highly suggestive of acute PCP. Eleven (58%) of the 19 patients with discordant PCR and CYT results had received prior anti-PCP prophylaxis. In this clinical setting in particular and in the evaluation of sputum specimens, the ability of PCR to detect a low parasitic load suggests that this technique may become an important additional tool, along with current cytological methods, for the detection of P. carinii.

By using a recently developed PCR-solution hybridization enzyme-linked assay (PCR-SHELA), we investigated Pneumocystis carinii in bronchoalveolar lavage fluid samples and induced sputa of patients with pneumocystosis. In detecting P. carinii, PCR-SHELA proved more sensitive than immunofluorescence staining or a single PCR and significantly more diagnostically specific than a nested PCR. Our data suggest that PCR-SHELA could be used to detect P. carinii organisms in respiratory samples, particularly in patients with uncertain diagnoses.

Despite recent declines in incidence, Pneumocystis carinii pneumonia (PCP) remains the most commonly occurring opportunistic illness among persons with AIDS in the United States. While P. carinii DNA has been detected in patient respiratory specimens and in air samples collected from various indoor environments housing PCP patients, the viability of these organisms is unknown. For this reason, we have developed and evaluated a molecular viability assay for P. carinii. This method is based upon the detection of P. carinii mRNA by a reverse transcription-PCR that employs specific primers from a member of the heat shock protein 70 family. Under optimal assay conditions, these primers were capable of detecting as few as 100 viable trophozoites as determined by ethidium bromide staining, while no signal was obtained from 106 trophozoites killed by heat, desiccation, or UV radiation. This assay was also capable of distinguishing P. carinii from other common fungi present in the air. Therefore, this molecular viability assay may be useful in conjunction with standard bioaerosol collection devices and procedures for the detection of viable P. carinii collected from various indoor environments. It may also be useful in confirming the presence of viable trophozoites in respiratory specimens collected by noninvasive techniques from putatively infected individuals.

The transmission of Pneumocystis carinii from person to person was studied by detecting P. carinii-specific DNA in prospectively obtained noninvasive deep-nasal-swab samples from a child with a documented P. carinii pneumonia (PCP), his mother, two contact health care workers, and 30 hospital staff members who did not enter the patient's room (controls). Nested-DNA amplification was done by using oligonucleotide primers designed for the gene encoding the mitochondrial large subunit rRNA of rat P. carinii (P. carinii f. sp. carinii) that amplifies all forms of P. carinii and internal primers specific for human P. carinii (f. sp. hominis). P. carinii f. sp. hominis DNA was detected in samples from the patient and all of his contacts versus none of the 30 hospital staff members. The results, as previously shown in murine models of P. carinii pneumonia, document that person-to-person transmission of P. carinii is possible. This observation suggests that immunocompromised patients not on PCP prophylaxis should not enter the room of a patient with PCP, and it also raises the question as to whether healthy contacts can transmit the disease to immunocompromised patients at risk.

Pneumocystis carinii pneumonia is a leading cause of morbidity and mortality in patients with the acquired immunodeficiency syndrome (AIDS). Much remains unknown about the basic biology of P. carinii and studies of this infection have been hampered by the lack of cultivation methods. We developed a sensitive and specific assay for P. carinii by utilizing DNA amplification of the P. carinii dihydrofolate reductase (DHFR) gene. By this method, P. carinii DNA was detected in the lungs of rats with experimentally induced P. carinii pneumonia 2 wk before the onset of histopathological changes. DNA amplification analysis of serum demonstrated that by 10 wk of corticosteroid treatment, 12 of 12 (100%) infected rats had circulating DHFR DNA. P. carinii DHFR DNA also was detected in the serum of patients with AIDS and active P. carinii pneumonia (12 of 14 sera collected prospectively). Patients with advanced AIDS but without a history of P. carinii pneumonia were negative by this assay (0 of 6 sera examined). Serum polymerase chain reaction may facilitate investigations into the natural history and epidemiology of P. carinii infection, provide insight into the pathogenesis of parasite dissemination, and offer a useful, noninvasive diagnostic test for the detection of human pneumocystosis.

The present study was conducted to determine the prevalence and significance of Pneumocystis carinii antigenemia in patients with acquired immunodeficiency syndrome (AIDS) and clinically or invasively diagnosed P. carinii pneumonitis. Single serum specimens from 20 AIDS patients invasively examined for P. carinii organisms and 106 AIDS patients with a clinical diagnosis only of P. carinii pneumonitis were blindly tested for P. carinii antigenemia by a counterimmunoelectrophoresis assay. In the 20 specimen-documented cases, the antigen test demonstrated a sensitivity of 75% and a specificity of 90%. The positive predictive value of the test was 90%, while the negative predictive value was 70%. In AIDS patients with specimen-documented P. carinii pneumonitis, the prevalence of P. carinii antigenemia coincided almost exactly with the prevalence of positive invasively obtained specimens (60 and 59%, respectively). In patients with a clinical diagnosis only of P. carinii pneumonitis, half as many (30%) were found to exhibit antigenemia. Sequential P. carinii antigen titers determined by a new latex agglutination technique on three AIDS patients with specimen-documented P. carinii pneumonitis demonstrated the influence of specific therapy upon P. carinii antigenemia and its potential prognostic application.

Early diagnosis of Pneumocystis carinii pneumonia, a life-threatening complication in immunosuppressed patients, may lower morbidity and mortality. We have developed a one-tube nested PCR assay for the detection of P. carinii in respiratory specimens. Four primers were selected from the sequence of the small-subunit rRNA gene of P. carinii to amplify a 265-bp fragment, and their specificities for P. carinii were confirmed by both theoretical evaluations (by computer-assisted comparison with the sequences in GenBank) and empirical evaluations (with DNA from medically important fungi and diagnostic samples). The assay was optimized for routine diagnostic use. Processing of the clinical samples is rapid and simple (digestion with proteinase K directly in PCR buffer at room temperature in the presence of 10% Chelex 100 and no further purification steps). Bovine serum albumin (1 mg/ml) and glycerol (10%) in the amplification buffer reduced the number of samples inhibitory to the PCR, as assessed by control reactions containing a size-modified target. A total of 749 clinical specimens (312 bronchoalveolar lavage, 403 sputum or induced sputum, and 34 other specimens) from 507 patients (295 human immunodeficiency virus [HIV]-infected and 164 non-HIV-infected patients and 48 patients whose HIV status was unknown) were tested by PCR, and the results were compared with those of an indirect immunofluorescence assay (IFA). Concordant results were obtained for 732 samples (646 negative and 86 positive). There were 17 discrepant results: 12 were PCR positive and IFA negative, and 5 were PCR negative and IFA positive. After resolution of the discrepant results by review of the patients' clinical data, the sensitivity and specificity were 94.8 and 99.1%, respectively, for PCR and 93.8 and 100%, respectively, for IFA. In conclusion, the short procedure time and the technical ease of this PCR assay render it suitable for implementation in routine diagnostic laboratories.

A rapid (time to completion, <4 h, including DNA extraction) and quantitative touch-down (QTD) real-time diagnostic Pneumocystis carinii PCR assay with an associated internal control was developed, using fluorescence resonance energy transfer (FRET) probes for detection. The touch-down procedure significantly increased the sensitivity of the assay compared to a non-touch-down procedure. Tenfold serial dilutions of a cloned target were used as standards for quantification. P. carinii DNA has been detected in respiratory specimens from patients with P. carinii pneumonia (PCP) and from patients without clinical evidence of PCP. The latter probably represents colonization or subclinical infection. It is logical to hypothesize that quantification might prove helpful in distinguishing between infected and colonized patients: the latter group would have lower copy numbers than PCP patients. A blinded retrospective study of 98 respiratory samples (49 lower respiratory tract specimens and 49 oral washes), from 51 patients with 24 episodes of PCP and 34 episodes of other respiratory disease, was conducted. PCR-positive samples from colonized patients contained a lower concentration of P. carinii DNA than samples from PCP patients: lower respiratory tract samples from PCP and non-PCP patients contained a median of 938 (range, 2.4 to 1,040,000) and 2.6 (range, 0.3 to 248) (P < 0.0004) copies per tube, respectively. Oral washes from PCP and non-PCP patients contained a median of 49 (range, 2.1 to 2,595) and 6.5 (range, 2.2 to 10) (P < 0.03) copies per tube, respectively. These data suggest that this QTD PCR assay can be used to determine if P. carinii is present in respiratory samples and to distinguish between colonization and infection.

A combination of three monoclonal antibodies, two prepared against human and one against rat Pneumocystis carinii, was used in an indirect fluorescent-antibody stain (IFA) to diagnose P. carinii in both bronchoalveolar lavage and lung biopsy specimens. This combination of monoclonal antibodies was specific for P. carinii and yielded bright fluorescence of both P. carinii cysts and trophozoites. A total of 126 specimens from 93 patients were stained for P. carinii by a toluidine blue O stain and the monoclonal IFA. Forty-five (35.7%) of these were positive for P. carinii by toluidine blue O, and 43 (34.1%) were positive by IFA. There was 98.4% agreement between both methods, with no false-positive and two false-negative occurrences by IFA. An IFA procedure with monoclonal antibodies such as those used in these studies can provide a simple, fast, and sensitive method for diagnosing P. carinii pneumonia by microbiology laboratories.

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There is a need to develop noninvasive methods for the diagnosis of Pneumocystis carinii pneumonia in patients unable to undergo bronchoscopy or induction sputum. Oral wash specimens are easily obtained, and P. ca- rinii nucleic acid can be amplified and demonstrated by PCR. In routine clinical use, easy sample processing and single-round PCR are needed to ensure rapid analysis and to reduce the risk of contamination. We developed a single-round Touchdown PCR (TD-PCR) protocol with the ability to detect PCR inhibition in the specimen. The TD-PCR was evaluated in a routine diagnostic laboratory and was compared to a previously described PCR protocol (mitochondrial RNA) run in a research laboratory. Both PCR methods amplified a sequence of the mitochondrial rRNA gene of P. carinii. Paired bronchoalveolar lavage (BAL) and oral wash specimens from 76 consecutive human immunodeficiency virus type 1-infected persons undergoing a diagnostic bronchoscopy were included. The TD-PCR procedure was quicker than the mitochondrial PCR procedure (<24 versus 48 h) and, compared to microscopy, had sensitivity, specificity, and positive and negative predictive values of 89, 94, 93, and 91%, respectively, for oral wash specimens and 100, 91, 90, and 100%, respectively, for BAL specimens. Our results suggest that oral wash specimens are a potential noninvasive method to obtain a diagnostic specimen during P. carinii pneumonia infection and that it can be applied in a routine diagnostic laboratory.

Quantitative PCR (qPCR) is more sensitive than microscopy for detecting Pneumocystis jirovecii in bronchoalveolar lavage (BAL) fluid. We therefore developed a qPCR assay and compared the results with those of a routine immunofluorescence assay (IFA) and clinical data. The assay included automated DNA extraction, amplification of the mitochondrial large-subunit rRNA gene and an internal control, and quantification of copy numbers with the help of a plasmid clone. We studied 353 consecutive BAL fluids obtained for investigation of unexplained fever and/or pneumonia in 287 immunocompromised patients. No qPCR inhibition was observed. Seventeen (5%) samples were both IFA and qPCR positive, 63 (18%) were IFA negative and qPCR positive, and 273 (77%) were both IFA and qPCR negative. The copy number was significantly higher for IFA-positive/qPCR-positive samples than for IFA-negative/qPCR-positive samples (4.2 ± 1.2 versus 1.1 ± 1.1 log10 copies/μl; P < 10−4). With IFA as the standard, the qPCR assay sensitivity was 100% for ≥2.6 log10 copies/μl and the specificity was 100% for ≥4 log10 copies/μl. Since qPCR results were not available at the time of decision-making, these findings did not trigger cotrimoxazole therapy. Patients with systemic inflammatory diseases and IFA-negative/qPCR-positive BAL fluid had a worse 1-year survival rate than those with IFA-negative/qPCR-negative results (P < 10−3), in contrast with solid-organ transplant recipients (P = 0.88) and patients with hematological malignancy (P = 0.26). Quantifying P. jirovecii DNA in BAL fluids independently of IFA positivity should be incorporated into the investigation of pneumonia in immunocompromised patients. The relevant threshold remains to be determined and may vary according to the underlying disease.

A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect <5 copies of a plasmid standard per tube. It was reproducibly quantitative (r = 0.99) over 6 log values for standards containing ≥5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer. In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical, such as in vitro antimicrobic susceptibility testing or in vivo immunopathological experiments.

Isoprinosine has been reported to decrease progression to AIDS, primarily by preventing Pneumocystis carinii pneumonia (PCP), in human immunodeficiency virus-infected patients, but the mechanism of action is unknown. para-Acetamidobenzoic acid (PAcBA), one component of isoprinosine, is structurally related to para-aminobenzoic acid (PABA), a precursor of de novo folate synthesis. This pathway is known to be important for P. carinii because sulfonamides, which are effective anti-P. carinii agents, inhibit incorporation of PABA into folate precursors by the enzyme dihydropteroate synthetase (DHPS). Inhibition of P. carinii DHPS by PAcBA was investigated by using two assays. In short-term cultures of P. carinii from rats, [3H]PABA incorporation into reduced folates was inhibited by both isoprinosine (mean +/- standard error 50% inhibitory concentration [IC50], 20 +/- 8.4 microM) and PAcBA free acid (IC50, 240 +/- 100 microM); a soluble PAcBA salt was more potent than PAcBA free acid alone (IC50, 29 +/- 48 microM). The activity of PAcBA free acid was confirmed in a cell-free DHPS inhibition assay (IC50, 120 +/- 120 microM). Inosine and dimethylaminopropanol, two other components of isoprinosine, were poor inhibitors of PABA incorporation (IC50, > 1,000 microM). PAcBA free acid also showed activity in inhibiting the DHPS of Toxoplasma gondii, but was a poor inhibitor of the DHPSs of Escherichia coli and Saccharomyces cerevisiae. In a rat model of PCP, the PAcBA salt administered intraperitoneally demonstrated no activity against established PCP either alone or when used in combination with trimethoprim; the lack of efficacy in this model may be due to the rapid metabolism of the drug. Prevention of PCP by PaCBA through inhibition of P. carinii DHPS may explain the activity of isoprinosine in decreasing the progression to AIDS in human immunodeficiency virus-infected patients.

Pneumocystis carinii is a eukaryotic microbe which causes fatal pneumonia in patients with AIDS. Oligonucleotide primers were used to amplify the 5S rDNA sequence of P. carinii by the polymerase chain reaction (PCR) in various clinical and animal samples. Of 35 independent lung specimens tested, PCR detected the P. carinii sequence in all 23 cases which were known to be P. carinii infected, i.e., 15 from mice, 1 from rat, 3 from human autopsy, and 4 from biopsy of AIDS patients by needle aspiration. The results were consistent with clinical and microscopic diagnosis. The detection was highly sensitive and specific. Direct sequencing of these amplified DNAs revealed homogeneity of 5S rDNA sequences of independent isolates from mice, rats, and humans. Preliminary trials manifested efficacy of the PCR method to detect P. carinii sequences in induced sputum or blood from AIDS patients, the latter case suggesting that P. carinii might enter peripheral blood via phagocytosis or direct intrusion. Development of less-invasive or noninvasive PCR diagnostic techniques to detect P. carinii infection would greatly facilitate therapeutic and prophylactic management of P. carinii pneumonia.

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Three HIV positive subjects presented with symptoms and radiographic changes suggestive of Pneumocystis carinii pneumonia. Methenamine silver staining of bronchoscopic alveolar lavage (BAL) fluid was negative (from one sample in one patient and two samples in the other two patients). Open lung biopsy was performed because of uncertain clinical progress and diagnosis; all three patients were found to have multiple pulmonary granulomata encasing numerous P carinii organisms. DNA amplification, using P carinii specific oligonucleotides, was performed on stored bronchoscopic BAL samples. P carinii specific amplification product was detected by ethidium bromide staining after electrophoretic separation on agarose gel in one case, and by the more sensitive technique of oligohybridisation in all three cases. In granulomatous P carinii pneumonia organisms are rarely identified in bronchoscopic alveolar lavage samples using histochemical staining, but are detectable by DNA amplification, although not at levels which can be readily distinguished from low, subclinical infection.

BACKGROUND—Pneumocystis carinii is an important pathogen in immunodeficiency but may be an unrecognised cause of respiratory compromise.
OBJECTIVES—To ascertain the incidence of P carinii pneumonia (PCP) at presentation of severe combined immunodeficiency (SCID), whether it had been diagnosed, and the effect of treatment on outcome.
SETTING—The supraregional paediatric bone marrow transplant unit for primary immunodeficiencies at Newcastle General Hospital.
METHODS—Retrospective case note review of infants referred with a diagnosis of SCID from 1992 to 1998.
RESULTS—Ten of 50 infants had PCP at presentation; only one was diagnosed before transfer. Eight were diagnosed by bronchoalveolar lavage and two by lung biopsy. In only one was P carinii identified in nasopharyngeal secretions. Five required ventilation for respiratory failure but all were successfully treated with co-trimoxazole and methylprednisolone with or without nebulised budesonide. Nine survived to bone marrow transplantation and four are long term survivors after bone marrow transplantation; no deaths were related to PCP.
CONCLUSIONS—PCP is a common presenting feature of SCID but is rarely recognised. Bronchoalveolar lavage or lung biopsy are needed for diagnosis. Treatment with co-trimoxazole is highly successful.



Four stains for the detection of Pneumocystis carinii in respiratory specimens were compared for sensitivity, specificity, preparation time, and ease of interpretation. One hundred specimens were collected. Of these, 50 were induced sputum specimens and 50 were bronchoalveolar lavage fluid. All specimens were stained with Diff-Quik (DQ) (a modified Giemsa stain), a quick silver stain, and direct and indirect immunofluorescence stains. A positive specimen was defined as any smear positive by two or more of the methods. Fifty-eight percent of specimens were positive. Seventy-four percent of the sputum specimens and 42% of the bronchoalveolar lavages were positive. The sensitivities for detection of P. carinii in sputum were 92% with silver stain, 97% with direct immunofluorescence assay (DFA), 97% with indirect immunofluorescence assay (IFA), and 92% with DQ. The sensitivities for detection in bronchoalveolar lavage were 86% with silver stain, 90% with DFA, 86% with IFA, and 81% with DQ. Preparation times varied from 90 min for the silver stain and IFA to 3 min for DQ. Costs of the tests varied from $1.50 per slide for DQ to $10.00 per slide for the silver stain and DFA. Reading times varied from 10 to 30 min for the silver stain and DQ to less than 5 min for the immunofluorescence assays. We conclude that all of these tests are viable options for the clinical laboratory, and the choice will be influenced by factors such as clinical volume, ability to batch specimens, and expertise of technological support. A reasonable option may be to use the quick and inexpensive DQ as a screening test and to confirm negative smears with a more sensitive assay.

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A polymerase chain reaction (PCR)-based assay was developed for the detection of Pneumocystis carinii DNA in induced sputum and bronchoscopic alveolar lavage samples. The primer pair was selected from the published sequence of the thymidylate synthase gene of P. carinii derived from infected rats. The amplified DNA fragment of 403 bp was detected by agarose gel electrophoresis and by Southern and slot blot hybridization. No positive reaction was seen with DNA from different microorganisms typically found in the respiratory tract. P. carinii DNA was demonstrated in 30 of 42 sputum samples from immunosuppressed patients, whereas 21 of 42 sputum samples were positive by indirect immunofluorescence (IFL). Among the 42 patients, 14 were receiving prophylactic chemotherapy. In that group, PCR detected P. carinii in nine sputum samples, whereas IFL detected P. carinii in only four sputum samples. A positive PCR result was also seen in 5 of 43 IFL-negative bronchoscopic alveolar lavage samples from patients with respiratory symptoms. The PCR assay detected 10 copies of the target DNA, which corresponds to 10(-18) g of the specific P. carinii sequence. The results indicate that PCR amplification in combination with DNA hybridization is specific and is a more sensitive diagnostic method than IFL for the detection of P. carinii.

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AIM--To prepare a monoclonal antibody (EB9) against the trophozoite form of Pneumocystis carinii and to test its efficacy for detecting infection with this organism. METHOD--The sensitivity and specificity of the EB9 antibody were assessed by comparing it with other conventional stains (Papanicolaou, Giemsa and Grocott) and 3F6 antibody in 33 bronchoalveolar lavage specimens from HIV positive patients suspected of having P carinii pneumonia. RESULTS--P carinii infection was detected in 15 of 33 patients. In 14 cases the organism was detected by two or more of the staining methods used; however, EB9 failed to detect infection in two cases which were positive by other staining techniques. In one case P carinii infection was detected by EB9 only. CONCLUSION--The results of this study suggest that P carinii infection in the lung may occur in two forms: the cyst form and the trophozoite form, which may explain the observed variation in response to treatment.

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